Epidemiologic, Virologic, and Host Genetic Factors of Norovirus Outbreaks in Long-term Care Facilities

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1 Clinical Infectious Diseases MAJOR ARTICLE Epidemiologic, Virologic, and Host Genetic Factors of Norovirus Outbreaks in Long-term Care Facilities Veronica P. Costantini, 1 Emilie M. Cooper, 1 Hope L. Hardaker, 2 Lore E. Lee, 2 Marieke Bierhoff, 1,3 Christianne Biggs, 2 Paul R. Cieslak, 2 Aron J. Hall, 1 and Jan Vinjé 1 1 Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; 2 Public Health Division of the Oregon Health Authority, Portland; and 3 Department of Internal Medicine, VU University Medical Center, Amsterdam, The Netherlands (See the Major Article by Kambhampati et al on pages ) Background. In the Unites States, long-term care facilities (LTCFs) are the most common setting for norovirus outbreaks. These outbreaks provide a unique opportunity to better characterize the viral and host characteristics of norovirus disease. Methods. We enrolled 43 LTCFs prospectively to study the epidemiology, virology, and genetic host factors of naturally occurring norovirus outbreaks. Acute and convalescent stool, serum, and saliva samples from cases, exposed and nonexposed controls were collected. Norovirus infection was confirmed using quantitative polymerase chain reaction testing of stool samples or 4-fold increase in serum antibody titers. The presence of histo-blood group antigens (secretor, ABO, and Lewis type) was determined in saliva. Results. Sixty-two cases, 34 exposed controls, and 18 nonexposed controls from 10 norovirus outbreaks were enrolled. Forty-six percent of acute, 27% of convalescent case, and 11% of control stool samples tested norovirus positive. Outbreak genotypes were GII.4 (Den Haag, n = 3; New Orleans, n = 4; and Sydney, n = 2) and GI.1 (n = 1). Viral load in GII.4 Sydney outbreaks was significantly higher than in outbreaks caused by other genotypes; cases and controls shed similar amounts of virus. Forty-seven percent of cases shed virus for 21 days. Symptomatic infections with GII.4 Den Haag and GII.4 New Orleans were detected among nonsecretor individuals. Conclusions. Almost half of all symptomatic individuals shed virus for at least 21 days. Viral load was highest in GII.4 viruses that most recently emerged; these viruses also infect the nonsecretor population. These findings will help to guide development of targeted prevention and control measures in the elderly. Keywords. norovirus; long-term care facilities; shedding; secretor status. Noroviruses are the leading cause of severe gastroenteritis worldwide [1]. An estimated million cases of norovirus disease occur in the United States every year [2]. Noroviruses cause 50% of acute gastroenteritis outbreaks in long-term care facilities (LTCFs) [3, 4]. During norovirus outbreaks, all-cause hospitalizations and deaths increase by approximately 10% relative to nonoutbreak periods [5]. Clinical symptoms usually start with a sudden onset of vomiting and diarrhea, lasting for up to 72 hours, with more severe outcomes observed in young children (<5 years) and older adults ( 65 years) [2]. Noroviruses (nonenveloped single-stranded, positive-sense RNA viruses) are divided into at least 6 genogroups (GI-GVI) and further subdivided into more than 38 genotypes based on phylogenetic analysis of the major capsid protein [6, 7]. GII.4 strains have been responsible for the majority of outbreaks, with a new GII.4 variant strain emerging every 2 4 years [8, 9]. Histo-blood group antigens (HBGAs) have been identified as putative attachment factors for norovirus [10]. Persons carrying a Received 5 May 2015; accepted 4 August 2015; published online 26 October Correspondence: V. P. Costantini, Division of Viral Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd, MS G-04, Atlanta, GA (vcostantini@cdc.gov). Clinical Infectious Diseases 2016;62(1):1 10 Published by Oxford University Press for the Infectious Diseases Society of America This work is written by (a) US Government employee(s) and is in the public domain in the US. DOI: /cid/civ747 functional fucosyltransferase 2 (encoded by the FUT2 gene) are termed secretors and express HBGAs on the gut epithelial cells, whereas homozygous individuals with 428G>A nonsense mutations at the FUT2 gene, called nonsecretors, are almost completely protected from norovirus infections [11 16]. Data from challenge studies in healthy adults have helped to clarify how the interactions between virus, host genetics, and immunology result in norovirus illness [17 19]. Since most norovirus outbreaks occur in the elderly, we prospectively investigated outbreaks in 43 LTCFs from November 2009 to January 2013 in order to determine the epidemiology and virology of the outbreaks and the associations among norovirus disease or infection, viral genotype, and HBGAs. MATERIALS AND METHODS Ethics Statement The institutional review boards of the Oregon State Public Health Division and the Centers for Disease Control and Prevention (CDC) approved the study. All residents and staff of LTCFs were eligible for inclusion, excluding those cognitively or decisionally impaired. Written informed consent was obtained from each participant. Participant Recruitment Prior to the onset of each winter norovirus season (December March), LTCFs in Oregon were recruited to participate in the Norovirus in Long-term Care Facilities CID 2016:62 (1 January) 1

2 study. A total of 43 LTCFs were enrolled from November 2009 to January Individual study participants were recruited both before the norovirus season and when outbreaks occurred. An outbreak was suspected to be caused by norovirus based on clinical and epidemiologic characteristics [20]. Enrolled participants were classified as cases and exposed and nonexposed controls based on symptomatology and contact with a sick person (Figure 1). Only outbreaks that were reported to local health departments within 3 days of the onset date of the first case, had at least 10 sick individuals, had 20 potentially exposed individuals, and were still ongoing at the time of notification were selected for inclusion in the study. See Supplementary data for detailed study design and methods. Data and Sample Collection Clinical data were obtained directly from participants using a standardized questionnaire. The severity of gastrointestinal symptoms was assessed using a score system based on the duration and frequency of diarrhea or vomiting, fever, and treatment (Supplementary Table). Saliva samples were collected on the day of symptom onset or exposure (day 0) from cases and exposed controls, respectively, and also on days 1, 14, and 21 to study the immune responses, which will be described in another manuscript. Serum and stool specimens were collected from cases and exposed controls on days 0 and 21 and stored at 20 C and 4 C, respectively. Single saliva, serum, and stool samples were collected from nonexposed controls within 7 days after onset of the outbreak. Norovirus Detection, Quantification, and Genotyping Norovirus RNA from 50 µl of 10% fecal suspension was detected using GI/GII TaqMan real-time quantitative polymerase chain reaction (RT-qPCR) [9]. The P2 domain was amplified by RT-PCR using oligonucleotide primer sets specific forgi.1 and GII.4 viruses detected in the study. Genotyping and phylogenetic analysis were performed using MEGA5 software [21] and norovirus reference sequences [6]. All stool samples were also tested for enteropathogens using xtag Gastrointestinal Pathogen Panel (Luminex Molecular Diagnostic). Secretor Status and HBGA Phenotyping Secretor genotype, defined by a single nucleotide polymorphism at position 428G>A in the FUT 2 gene, was determined by pyrosequencing as described previously [22]. HBGA phenotypes were determined from saliva by enzyme immunoassay using Figure 1. Flow diagram of the study experimental design. From 2009 through 2013, 43 long-term care facilities were enrolled, and 10 norovirus outbreaks that qualified for inclusion in the study were reported. A total of 62 cases, 34 exposed controls, and 18 nonexposed controls were enrolled based on the presence of clinical symptoms and exposure to a case. a The immune responses will be described in another article. 2 CID 2016:62 (1 January) Costantini et al

3 monoclonal antibodies (1 µg/ml) against blood group antigens A or B (Ortho Clinical Diagnostics), H antigen, Lewis (Le) a, Lewis b, Lewis x (Santa Cruz Biotechnology), or Lewis y (Calbiochem) [23]. Three saliva samples from each participant were tested for HBGA phenotypes. To determine whether norovirus strains could bind saliva from secretor and nonsecretor individuals, 100 µl of GII.4 Den Haag, GII.4 New Orleans, or GII.4 Sydney virus-like particles (VLPs) at 0.5, 1.0, and 5.0 µg/ml were added to saliva-coated wells. VLP and saliva were genotype matched by outbreak. VLP binding was detected with polyclonal antibody against the norovirus strain and 3,3,5,5 tetramethylbenzidine substrate. Norovirus GII.4 Den Haag and GII.4 Sydney VLPs were kindly provided by Dr Charles Arntzen (Arizona State University) and Dr Ralph Baric (University of North Carolina), respectively. Data Analysis Nonparametric data were compared using Wilcoxon rank sum, and categorical variables were analyzed using Fisher exact test. Log transformation and 1-way analysis of variance followed by Tukey multiple comparison test was applied to viral loads to compare mean values. Spearman correlation was used to compare illness and shedding duration with severity score. Shedding duration was analyzed as Kaplan Meier survival probability. GraphPad Prism 5 (Graph PadSoftware Inc., California) was used to perform analyses, and P values <.05 were considered statistically significant. RESULTS Demographics From November 2009 through January 2013, 39 norovirus outbreaks were reported at 30 (70%) of 43 LTCFs (Supplementary Figure). Ten (26%) of 39 outbreaks met the inclusion criteria of our study and resulted in 386 illnesses, 29 hospitalizations, and 5 associated deaths. Sixty-two cases (65% aged 70 years), 34 exposed controls (9% aged 70 years), and 18 nonexposed controls (5% aged 70 years) were enrolled (Table 1). Most cases were LTCF residents (79%), whereas the exposed control group predominantly consisted of staff who provided direct patient care (68%), and 50% of the nonexposed controls were nonpatient care staff. Symptoms included diarrhea (84%), fatigue (81%), vomiting (76%), and nausea (74%). Presence of both vomiting and diarrhea (62%) was reported more frequently than either symptom alone (14% and 22%, respectively) (Table 2). Adults aged 70 yearsweremorelikelythanyoungerpatientstoreportboth symptoms (73% vs 45%; P =.035). Two (3%) of 62 cases reported to have had a similar illness within the preceding year. Illness duration was longer in cases aged 70 years (n = 29; median, 4; interquartile range [IQR], 3 4) than aged <70 years (P =.041), with 19 (60%) lasting >3 days and 4 (13%) lasting >5 days (Figure 2A). The median severity score was 5.0 (n = 62; Table 1. Characteristic Demographic Characteristics of Study Participants Cases (N = 62) Exposed Controls (N = 34) Nonexposed Controls (N = 18) No. (%) No. (%) No. (%) Sex Male Female Race Black White Asian Hispanic Native American Unknown Age, y Median [min, max] 81 [25 95] 44 [22 85] 49 [21 88] Group class Resident Patient care staff Nonpatient care staff Household members range, 2 10). The median score was higher in those aged and years (median, 6) than in those aged and 90 years (median, 5; Figure 2B). No correlation was observed among severity score, illness, or shedding duration for the group or after age stratification (n = 29; Figure 2C). Norovirus Detection and Genotyping The 10 studied outbreaks occurred in 7 LTCFs, with more than 1 outbreak detected in 2 LTCFs (Table 3). Norovirus was detected in stool samples from 42 cases, 4 exposed controls, and 1 nonexposed control. No sequence variation among samples from the same outbreak was detected except for outbreaks A, F, and H. Phylogenetic analysis of P2 sequences (Figure 3) showed that 18 (95%) of the 19 cases in outbreak A were typed as GII.4 New Orleans and 1 case as GII.4 Den Haag. Positive samples from exposed and nonexposed controls (A_268_Exp_Cv and A_205_NonExc, respectively) as well as convalescent stool samples had 100% sequence homology with the outbreak strain. One (H_552) of 3 samples from outbreak H (GII.4 New Orleans) had 1 amino acid substitution in the P2 sequence. Sequences from the same participant at days 0 and 21 were identical. Three of the 4 samples from outbreak F genotyped as GI.1, while the 4th sample (F_446_Ac) was typed as GII.4 New Orleans. However, since the convalescent stool sample from F_446 was also typed as GI.1, the GII.4 New Orleans virus at day 1 was considered unrelated to the outbreak. Norovirus in Long-term Care Facilities CID 2016:62 (1 January) 3

4 Table 2. Frequency of Symptoms, Medical Care, and Treatment Among Cases (N = 62) All Ages (N = 62) y (N = 9) y (N = 13) y (N = 29) 90 y (N = 11) Variable No. % No. % No. % No. % No. % Symptoms Vomiting Duration (days) > Episode/day > Diarrhea Duration (days) > Episode/day > Vomiting and diarrhea Blood in stools Fever Shaking chills Myalgia Headache Abdominal pain Nausea Fatigue Illness duration (days) a Mean [95% confidence interval] 3.5 [ ] 2.8 [ ] 3.3 [ ] 3.8 [ ] 4.1 [ ] Median [interquartile range; range] 4 [3 4;1 8] 3 [2 3.75;1 8] 3.5 [2.25 4;1 6] 4 [3 4;1 8] 4 [4;3 6] Medical care None Emergency department Hospitalization Treatment Oral rehydration Intravenous fluids Perceived illness severity Mild Moderate Severe Last similar illness b <2 wk mo >1 y Never Unknown a Information available from 49/62 cases (age years [n = 8], years [n = 12], years [n = 21], and 90 years [n = 8]). b Last time as reported by the participant. Virus Shedding and Coinfections Among 179 stool samples, 104 (55 acute, 49 convalescent, 47 acute/convalescent pairs) were collected from 62 cases (Table 3). Forty-two (76%) of 55 acute and 20 (42%) of 49 convalescent samples tested positive for norovirus. Six acute GII.4 New Orleans positive stool samples also tested positive for Clostridium difficile toxin A/B (n = 5) and Giardia (n = 1). 4 CID 2016:62 (1 January) Costantini et al

5 Figure 2. Duration of illness, virus shedding, and severity score in cases reported during the study. A, Illness duration data from 49 cases were based on questionnaire responses. Graph represents median + interquartile range (IQR) for each age group. B, The severity of norovirus disease was assessed for all cases (n = 62) on the basis of a score system; scores ranged from 0 to 17, with higher scores indicating more severe disease (Supplementary Table 1). Graph represents median + IQR for each age group. C, Illness duration, virus shedding duration, and severity score were obtained from 29 cases from which both acute and convalescent stool samples were collected. Boxes represent 25th percentile, median, and 75th percentile, and the whiskers show the 10th 90th percentile for illness duration (white boxes), virus shedding duration (gray boxes), and severity score (stripe boxes). A total of 59 stool samples (29 acute, 30 convalescent, 26 acute/convalescent pairs) were collected from 34 exposed controls. Of these, 3 (10%) of 29 acute and 1 (3%) of 30 convalescent tested positive for norovirus. One (6%) of 16 stool samples from nonexposed controls tested positive for norovirus. The median collection date after onset of symptoms for acute positive stool was 3 days (n = 42; range, 0 8), with no significant difference among genotypes (P =.732). No significant differences were found between the mean acute viral loads in samples from cases in outbreaks with the same genotype (Table 3). In contrast, compared with outbreaks caused by GII.4 New Orleans (P =.029), GII.4 Den Haag (P =.009), and GI.1 Norwalk (P =.0281), the mean viral load in GII.4 Sydney outbreaks ( copies/g stool; range, copies/g stool) was significantly higher (Figure 4). Similarly, the mean viral load in GII.4 New Orleans outbreaks ( copies/g stool; range, copies/g stool) was significantly higher than in GII.4 Den Haag (P =.008) and GI.1 Norwalk (P =.0065) outbreaks. No difference was found between mean viral load from GI.1 Norwalk and GII.4 Den Haag outbreaks (P =.4695). Mean acute viral shedding was comparable in cases (n = 40; copies/g stool) and asymptomatic exposed controls (n = 3; copies/g stool; P =.4611). No correlation was observed between viral load and age (r = ; P =.2031). Prolonged shedding ( 21 days) was detected in 16 (47%) of the 35 cases with positive acute stool. The mean acute viral load ( copies/g stool) among cases with prolonged shedding was not significantly different from those that had a negative convalescent stool ( copies/g stool; P =.1012; Figure 5A). Nevertheless, cases with acute titers copies/g stool (n = 13) were significantly more likely to remain positive Norovirus in Long-term Care Facilities CID 2016:62 (1 January) 5

6 Table 3. Summary of Diagnostics and Genotyping of 10 Norovirus Outbreaks in Long-term Care Facilities Outbreak Long-term Care Facility Date Group No. Participant Enrolled Stool Samples No. Positive/No. Collected (%) Viral Load a (RNA Copies/g Stool) Acute Convalescent Acute Convalescent Genotype b A v March 2010 Cases c,d /22 (86) 8 c /19 e (42) GII.4 New Exposed controls 5 0/3 (0) 1/4 (25) Orleans Nonexposed controls 11 1/10 (10) 0/0 (0) B viii May 2010 Cases 6 3/4 (75) 1/3 (33) GII.4 Den Haag Exposed controls 4 1/3 (33) 0/2 (0) Nonexposed controls 0 0/0 (0) 0/0 (0) - - C xviii January 2011 Cases 6 4 f /6 g (67) 0/4 (0) GII.4 New Exposed controls 5 0/5 (0) 0/5 (0) - - Orleans Nonexposed controls 0 0/0 (0) 0/0 (0) - - D xi April 2011 Cases 5 2/4 (50) 1/3 (33) GII.4 Den Haag Exposed controls 7 0/6 (0) 0/6 (0) - - Nonexposed controls 2 0/2 (0) 0/0 (0) - - E v September Cases 2 0/2 (0) 1/2 (50) GII.4 Den Haag 2011 Exposed controls 1 0/1 (0) 0/1 (0) - - Nonexposed controls 0 0/0 (0) 0/0 (0) - - F xv November Cases 5 4/5 c (80) 2/5 (40) GI.1Norwalk 2011 Exposed controls 2 0/2 (0) 0/2 (0) - - Nonexposed controls 2 0/1 (0) 0/0 (0) - - G v April 2012 Cases 6 4 d /4 (100) 3/5 (60) GII.4 New Exposed controls 2 1/1 (100) 0/2 (0) Orleans Nonexposed controls 1 0/1 (0) 0/0 (0) - - H xix April 2012 Cases 4 4 d /4 (100) 2/4 e (50) GII.4 New Exposed controls 1 0/1 (0) 0/1 (0) - - Orleans Nonexposed controls 2 0/2 (0) 0/0 (0) - - I xvi November Cases 3 1/3 (33) 1/3 (33) GII.4 Sydney 2012 Exposed controls 4 1/4 (25) 0/4 (0) Nonexposed controls 0 0/0 (0) 0/0 (0) - - J xv January 2013 Cases 1 1/1 (100) 1/1 (100) GII.4 Sydney Exposed controls 3 0/3 (0) 0/3 (0) - - Nonexposed controls 0 0/0 (0) 0/0 (0) - - Total /100 (46) 21/79 (27) a Mean. b Outbreak genotype was established based on P2 region from positive stool samples collected from cases. c At least 1 sample has a different genotype from that of the outbreak strain. d Coinfection with Clostridium difficile toxin A/B was detected in 3, 1, and 1 acute samples (outbreak A, G, and H, respectively). e Clostridium difficile toxin A/B was detected in 1 convalescent norovirus-negative stool sample. f Coinfection with Giardia was detected in 1 sample. g Clostridium difficile toxin A/B was detected in 1 acute norovirus-negative stool sample. compared with those shedding <10 10 copies/g stool (P <.001), with 9 (70%) lasting for at least 4 weeks (Figure 5B). HBGAs and Disease Of 114 individuals, 103 (90.4%) were secretors, of which 42 (40.4%) were homozygous and 61 (59.6%) were heterozygous. To investigate potential associations between HBGAs and norovirus infection or disease, the ABO, Lewis, and secretor status distributions among cases and exposed controls (n = 96) were stratified by infection (infected or noninfected) and disease (symptomatic or nonsymptomatic) and compared for each genotype or variant. Eighty-nine (93%) of 96 participants were secretor (Le a b+ ). All ABO types were detected among cases and exposed controls. Among 7 nonsecretors, 5 were Le a+b (Lewis positive) and 2 were Le a b (Lewis negative). The 18 nonexposed controls included both secretors (n = 14; ALe a b+ n=7;ale a b n=1andole a b+, n = 6) and nonsecretors (n = 4, Le a+b ). The blood type A/secretors (n = 4) of the GI.1 outbreak were symptomatic. Fifty-five (94.8%) of 58 symptomatic individuals in GII.4 outbreaks were secretors. However, secretors were as likely as nonsecretors to be infected. In GII.4 Den Haag outbreaks, 15 (60%) of 25 secretors were infected compared with 1 (50%) of 2 nonsecretors (P = 1.0). Similarly, 39 (80%) of 49 secretors and 2 (66%) of 3 nonsecretors were infected with 6 CID 2016:62 (1 January) Costantini et al

7 Figure 3. Phylogenetic analysis of (A) norovirus GI strains (outbreak F) and (B) norovirus GII strains (outbreaks A E and G J) based on amino acid sequences of the P2 domain of major capsid protein. Samples are listed by outbreak ID, followed by participant ID and collection time (Ac, acute; Cv, convalescent). Reference sequences are labeled by their name. The evolutionary history was inferred by using the maximum likelihood method based on the JTT matrix-based model. The tree with the highest log likelihood is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JDT model and then selecting the topology with superior log likelihood value [24]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA5 [21]. All sequences from cases, except where indicated. GenBank accession numbers of reference sequences are as follows: GI.1 Norwalk (M87661), GI.2 Southampton (L07418), GI.3 Desert Shield (U04469), GI.3b Stavanger (AF145709), GI.4 Chiba (AB042808), GI.5 Musgrove (AJ277614), GI.6 Hesse (AJ277615), GI.7 Winchester (AJ277609), GI.8 Boxer (AF538679), GI.9 Vancouver 730 (HQ637267), GII.4 Bristol (X76716), GII.4 Richmond (GU ), GII.4 US95/96 (AJ ), GII.4 Farmington Hills 2002 US (AY ), GII.4 Hunter 2004 AUS (AY ), GII.4 Yerseke 2006a NLD (EF ), GII.4 Den Haag 2006b NLD (EF ), GII.4 Apeldoorn_2007 (AB445395), GII.4 New Orleans 2009 (GU445325), and GII.4 Sydney 2012 (JX459908). Abbreviations: ExC, exposed control; non-exc, nonexposed control. GII.4 New Orleans viruses (P =.520), whereas GII.4 Sydney infected 5 (55%) of 9 secretors, but none of 2 nonsecretors (P =.455). In GII.4 outbreaks, we found that individuals regardless of blood type were infected, regardless of the GII.4 variant. In summary, no association between secretor status or blood type and symptomatic infection was found for GII.4 viruses. Three of 7 nonsecretors were infected by either GII.4 Den Haag (n = 1; Le a+b ) or GII.4 New Orleans (n = 2; Le a+b and Le a b ). To determine if nonsecretor individuals could be infected, saliva samples from all participants were incubated with different GII.4 VLPs (Figure 6). All VLPs bound to saliva samples from secretors. GII.4 Den Haag VLPs also bound to saliva samples from nonsecretors (n = 3, median, 0.432, range, ; Figure 6A). In contrast, GII.4 New Orleans and Sydney VLPs were able to bind saliva from nonsecretors Le a+b, but not from Le a b (Lewis negative), when the VLP concentration was increased to 5.0 µg/ml. Five of 6 Le a+b saliva samples were from controls, suggesting that lack of infection in these individuals could be the result of exposure to small amounts of virus. VLPs did not bind to saliva from either an exposed control (I_561) or a case (A_309) who were nonsecretor Le negative (Le a b ). DISCUSSION In the United States, the number of individuals aged >65 years is expected to grow to 79.7 million (21% of the population) by 2040 [25], of which a substantial number will live in LTCFs. Since more than 60% of reported norovirus outbreaks occur in the elderly population [4], targeted vaccination of high-risk groups such as the elderly may significantly reduce the burden of hospitalization and death. During our 3-year study, 70% of the prospectively enrolled LTCFs reported at least 1 norovirus outbreak. No associations between severity of disease, illness duration, and virus shedding was found. Viral loads were as Norovirus in Long-term Care Facilities CID 2016:62 (1 January) 7

8 Figure 4. Viral shedding during norovirus outbreaks reported during the study. Box plots represent log 10 viral shedding (median and 25th and 75th quartiles [box], 10th and 90th percentiles [whiskers]). Data were stratified for cases/exposed controls/nonexposed controls, during acute (day 0 8) or convalescent (day 17 32) time with the same genotype. Only positive samples are included; therefore, lack of value indicates negative samples or sample not collected (Table 3). Classification was based on virus genotype detected in stool sample. Viral shedding was log 10 transform and analysis of variance 1-way followed by Tukey multiple comparison test was applied to compare all mean values (P <.05). Abbreviations: Ac, acute; Cv, convalescent; ExC, exposed control; non-exc, nonexposed control. high as >10 13 copies/g stool and were significantly higher in outbreaks caused by GII.4 New Orleans and GII.4 Sydney viruses shortly after these variants had emerged [9, 26]. Symptomatic and asymptomatic individuals shed similar amounts of virus regardless of duration of shedding. Almost 50% of the cases shed virus for at least 3 weeks in high titers, suggesting that Figure 5. Shedding duration among cases. Acute (day 0 8) and convalescent stools sample (day 17 32) were collected from 35 cases. A, Norovirus was detected in 16/35 convalescent samples. Data represent mean viral load (copies/g stool) plus standard deviation. Short shedding convalescent samples were negative for norovirus. B, Proportion of positive samples stratified by norovirus concentration in acute stool samples. The survival distributions were significantly different (log rank test, χ 2 = 12.45, P <.05). Cases with an initial shedding higher than copies per gram stool were significantly more likely to continue shedding virus for 3 4 weeks (P <.001). 8 CID 2016:62 (1 January) Costantini et al

9 Figure 6. Binding of norovirus virus-like particle (VLP) to saliva samples collected during the study. Saliva samples were assayed for their ability to bind outbreak-specific norovirus VLPs. A, Norovirus VLP (0.5 µg/ml) binding to saliva samples collected from secretors (n = 103) and nonsecretors (n = 11) during norovirus outbreaks reported during the study. Triangles represent corrected optical density (OD) (450 nm) from individual saliva samples stratified for secretor status and outbreak-specific norovirus genotype. All cases/exposed controls/nonexposed controls were included. Line represents median + interquartile range. Saliva from secretors and nonsecretors were able to bind GII.4 Den Haag, but not GII.4 New Orleans or GII.4 Sydney VLPs. B, Saliva samples from nonsecretors (negative on A) were assayed for their ability to bind increasing amounts of genotype-specific norovirus VLPs (0.5, 1.0, 5.0 µg/ml). Significant differences were observed after incubation of 1.0 µg/ml or 5.0 µg/ml with saliva samples from Le a+b (Lewis positive) (P <.0001), but not from Le a b (Lewis negative). Abbreviations: Ca, case; ExC, exposed control; non-exc, nonexposed control. some individuals are able to clear norovirus infection more rapidly than others. Our data showed that nonsecretor individuals could also be infected by GII.4 Den Haag and GII.4 New Orleans viruses. This observation was further supported by confirmation that saliva from nonsecretor Le a+b individuals did bind to GII.4 Den Haag and GII.4 New Orleans VLPs. An interesting observation in our study was the fact that although individual virus loads were similar or higher (range, genomic copies/g of stool) than noted in previous reports [18, 19, 27 30], the highest viral loads correlated with the recent emergence of new GII.4 variants. Viral load in acute asymptomatic individuals (exposed controls) was also relatively higher for GII.4 Sydney. Because outbreaks with higher viral load occurred relatively soon after the emergence of GII.4 New Orleans in 2009 and GII.4 Sydney in 2012, increased viral load in both symptomatic and asymptomatic individuals may suggest more efficient virus replication. After recovery from clinical symptoms, infected individuals can still shed virus for weeks [18, 19, 30, 31]. In our study, 47% of cases who provided convalescent stool were positive at days The mean viral load in convalescent samples (10 8 copies/g stool) was much higher than reported in other studies (10 4 /g), suggesting that shedding could extend for several more weeks in these populations [32]. Although the viral load in convalescent samples was significantly higher than the minimum infectious dose [33, 34], reports from nosocomial norovirus outbreaks showed that primarily symptomatic patients contributed to transmission of disease [28, 29]. Akeyfinding of our study was that more recent GII.4 variants can infect nonsecretors as well as secretors. Nonsecretor individuals have near-complete resistance to GI.1 and GII.4 infection; however, with lower risk, they can also be infected by GI.3 and GII.2 strains [11, 13, 15, 17]. We found that saliva from Le a+b nonsecretors bind GII.4 Den Haag, GII.4 New Orleans, and GII.4 Sydney VLPs, supporting the concept that more recent GII.4 variants are able to bind glycans from Lewis-positive nonsecretor individuals [35]. This may explain the increased number of outbreaks when GII.4 Den Haag emerged in In contrast, no increase in outbreak activity was observed when GII.4 New Orleans and GII.4 Sydney emerged, which may be a consequence of similar affinity for HBGAs [36, 37]. We also found a Lewis-negative (Le a b ) individual who was symptomatically infected, in contrast to other reports [12, 14]. These differences could be linked to different genotypes detected in each study or the presence of an HBGA-like substance on the surface of the gut that provides noroviruses with the opportunity to infect intestinal cells [38, 39]. Our study had several limitations. First, lack of norovirus detection in stool samples from several cases may have been due to late collection, intermittent shedding, or shedding below the detection level. Our ability to detect significant associations between secretor status and infection/disease was limited by small numbers of nonsecretors and by small numbers of participants affected by GII.4 Sydney outbreaks. Individual behavior and different exposure scenarios may have influenced participant classification as cases or controls, since healthcare workers were probably consciously more careful than residents when in contact with a sick person. In summary, we prospectively studied 10 norovirus outbreaks in LTCFs and confirmed that infected people can asymptomatically shed virus at high levels for at least 3 weeks. Although secretor Norovirus in Long-term Care Facilities CID 2016:62 (1 January) 9

10 status is considered an important factor of innate protective immunity, the most recent GII.4 strains appear to be able to infect the nonsecretor population as well. Future studies are needed to better understand whether protection or susceptibility is related to immune response [40]. Whether the shedding of viruses after 3 weeks represents infectious virus is another key question that will need to be answered. Such studies will help us to better understand the dynamics of norovirus outbreaks in the elderly and guide development of targeted prevention and control measures. Supplementary Data Supplementary materials are available at Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. Notes Acknowledgments. We are grateful to Matthew Jaqua, John Oh, Rachele Cruz, Sandra Martin, Keenan Williamson, Emilio Debess, LaDonna Grenz, and Marjorie Yungclas of the Oregon Health Authority and Catherine Yen of the Centers for Disease Control and Prevention (CDC) for coordinating enrollment and data/sample collection. We also thank all residents and personnel at the long-term care facilities for their participation in this study. Disclaimer. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the CDC. Financial support. This work was supported, in part, by the National Institute of Food and Agriculture at the US Department of Agriculture (grant number ) and a grant to the CDC Foundation from Takeda Pharmaceuticals. Potential conflicts of interest. P.R.C.,L.E.L.,andH.L.H.report grants and nonfinancial support from the CDC Foundation. All other authors report no potential conflicts. 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